Obesity has emerged as the leading cause of mortality in the United States and there is a dearth of successful pharmacological therapies to treat it. Inhibition of fat processing in the digestive tract is a validated approach for anti-obesity treatment but there are significant problems with the existing therapeutics. The FDA-approved lipase inhibitor Orlistat prevents fat processing in the gut, leading to reduced body mass index in obese patients. The unprocessed fats are not absorbed by the body and pass through the digestive tract. While providing a critical proof-of-concept in humans, Orlistat suffers from low patient compliance because the unprocessed fats cause gastrointestinal side effects such as fecal urgency and oily spotting. Genetic and pharmacological evidence suggests that new drugs that inhibit fat processing steps downstream of lipase activity are expected to maintain the positive anti-obesity effects of lipase inhibition without the gastrointestinal side effects. These downstream steps occur in intestinal enterocytes, which process fats into triglyceride-rich particles for distribution in the body. Therefore we propose to develop a novel high-throughput phenotypic screen to identify small molecule inhibitors that act on fat processing in enterocytes. In Specific Aim 1, an enzyme-linked immunosorbance assay (ELISA) will be developed as a high throughput phenotypic screen to measure ApoB-48 secretion from Caco-2 human enterocyte cells. ApoB-48 is a core component of the triglyceride-rich particles secreted as the last step in fat processing by enterocytes. This screen will therefore detect compounds that act on any fat processing step downstream of lipase activity. In Specific Aim 2, a reference compound library will be used to demonstrate that the assay developed in Specific Aim 1 is capable of identifying relevant actives and to optimize high throughput screening conditions. The reference library will include compounds expected or known to inhibit fat processing in enterocytes. Successful completion of these Specific Aims will enable a Phase II SBIR program in which a large library of small molecules will be screened for inhibition of fat processing in enterocytes. Active compounds will be characterized in vitro in mechanism of action studies and in vivo in diet-induced obesity models. Mechanism of action studies may lead to the discovery of novel, druggable targets within this critical pathway. Thus the anticipated outputs of the Phase II study are promising anti-obesity lead compounds, novel targets, or both. PUBLIC HEALTH RELEVANCE: Despite significant public health and pharmaceutical industry efforts, obesity remains a formidable and unresolved epidemic. Obesity is estimated to contribute to over 300,000 deaths per year in the United States and costs the United States healthcare system $90 billion annually. Obese individuals are at heightened risk for a myriad of diseases, including diabetes, hypertension, stroke, cardiovascular disease, and cancer. The objective of this research is to discover new drugs to treat obesity by developing a high throughput phenotypic screen for the inhibition of fat processing in the intestine.